Positive identification of phospho-proteins using motif-specific, context-independent antibodies coupled with database searching

Abstract

The invention provides a method for producing antibodies that selectively recognize short, modified amino acid motifs substantially independent of the surrounding amino acid context in which the motif occurs. A novel class of motif-specific, context-independent antibodies is also provided. The invention encompasses modified motifs consisting of single modified amino acids, for example phosphotyrosine or acetylated lysine, as well other modified motifs of multiple amino acids, such as kinase consensus substrate motifs and protein-protein binding motifs relevant to cell signal transduction. Also provided are methods of profiling large and diverse protein populations on a genome-wide basis by utilizing the antibodies of the invention, and methods for the positive identification of cellular phosphoproteins using one or more motif-specific, context-independent antibodies of the invention coupled with protein database searching.

What is claimed is:  
     
         1 . A method for the positive identification of cellular phosphoproteins using motif-specific, context-independent antibodies coupled with database searching said method comprising the steps of: 
 (a) fractionating a preparation comprising a plurality of phosphoproteins on the basis of mass and/or isoelectric point (PI);    (b) contacting the fractionated preparation of step (a) with at least one motif-specific, context-independent antibody that binds a phosphorylated motif of known sequence, thereby to detect phosphoproteins comprising said motif; and    (c) utilizing a search program to search one or more protein database(s) for reported proteins having a sequence comprising said motif and having a mass and/or PI substantially matching that determined in step (a), thereby to positively identify at least one cellular phosphoprotein in said preparation as a reported protein of known sequence and mass and/or PI.    
     
     
         2 . The method of  claim 1 , wherein the fractionation of step (a) comprises gel electrophoresis.  
     
     
         3 . The method of  claim 1 , wherein the fractionation of step (a) comprises 2-dimensional gel electrophoresis separation on the basis of both mass and isoelectric point (PI).  
     
     
         4 . The method of  claim 1 , wherein said search program of step (c) comprises Scansite and/or Prosite search software.  
     
     
         5 . The method of  claim 1 , wherein said preparation comprises a crude cell extract or a partially purified cell extract.  
     
     
         6 . The method of  claim 5 , wherein said preparation is obtained from a diseased cell or tissue or from a cell or tissue susceptible to disease.  
     
     
         7 . The method of  claim 6 , wherein said disease is cancer.  
     
     
         8 . The method of  claim 1 , wherein said preparation is obtained from a cell, tissue or organism treated with a test compound.  
     
     
         9 . The method of  claim 8 , wherein said test compound is a kinase inhibitor.  
     
     
         10 . The method of  claim 1 , further comprising the step of (d) verifying the identity of said phosphoprotein as determined in step (c) by contacting said phosphoprotein with at least one antibody specific for said protein in phosphorylated or unphosphorylated form, thereby verifying the protein's identity.  
     
     
         11 . The method of  claim 1 , wherein a plurality of motif-specific, context-independent antibodies, each recognizing a different phosphorylated motif, is contacted with said fractionated preparation in step (b), and wherein the search of step (c) is for reported proteins comprising two or more of the different motifs.  
     
     
         12 . The method of  claim 1 , wherein said motif-specific, context-independent antibody binds a motif comprising a kinase consensus substrate motif or a protein-protein binding motif.  
     
     
         13 . The method of  claim 12 , wherein said motif comprises one or more phosphorylated amino acids.  
     
     
         14 . The method of  claim 12 , wherein said kinase consensus substrate motif is selected from the group consisting of a MAPK consensus substrate motif, a CDK consensus substrate motif, a PKA consensus substrate motif, a bulky ring-directed kinase consensus substrate motif, and an Akt consensus substrate motif, and wherein said protein-protein binding motif comprises a 14-3-3 binding motif or a PDK1 docking motif.  
     
     
         15 . The method of  claim 1 , wherein the precise target sequence preferably bound by the antibody employed in step (b) has first been determined, wherein the phosphoproteins detected in step (b) comprise said precise target sequence, and wherein the search of step (c) is for reported proteins having a sequence comprising said precise target sequence.  
     
     
         16 . The method of  claim 1 , wherein the fractionation of step (a) comprises immunoprecipitation.  
     
     
         17 . A method for the positive identification of cellular phosphoproteins using motif-specific, context-independent antibodies coupled with database searching, said method comprising the steps of: 
 (a) fractionating a cellular preparation comprising a plurality of phosphoproteins on the basis of mass and/or PI;    (b) contacting the fractionated preparation of step (a) with a plurality of motif-specific, context-independent antibodies that each bind a different phosphorylated motif of known sequence, thereby to detect at least one phosphoprotein comprising two or more of the different motifs; and    (c) utilizing a search program to search one or more protein database(s) for reported proteins having a sequence comprising the two or more different motifs detected in step (b) and having a mass and/or PI substantially matching that determined in step (a), thereby to positively identify at least one cellular phosphoprotein in said preparation as a reported protein of known sequence and mass and/or PI.    
     
     
         18 . The method of  claim 17 , wherein said preparation comprises a crude cell extract or a partially purified cell extract.  
     
     
         19 . The method of  claim 17 , wherein said motif-specific, context-independent antibody binds a motif comprising a kinase consensus substrate motif or a protein-protein binding motif.  
     
     
         20 . The method of  claim 17 , further comprising the step of (d) verifying the identity of said phosphoprotein as determined in step (c) by contacting said phosphoprotein with at least one antibody specific for said protein in phosphorylated or unphosphorylated form, thereby verifying the protein's identity.  
     
     
         21 . The method of  claim 17 , further comprising the step of (d) verifying the identity of said phosphoprotein as determined in step (c) by immuno-depletion of said phosphoprotein using at least one antibody specific for said protein in phosphorylated or unphosphorylated form.  
     
     
         22 . A method for the positive identification of cellular phosphoproteins using motif-specific, context-independent antibodies coupled with database searching, said method comprising the steps of: 
 (a) fractionating a cellular preparation comprising a plurality of phosphoproteins on the basis of mass and/or isoelectric point (PI);    (b) contacting the fractionated preparation of step (a) with a plurality of motif-specific, context-independent antibodies that each bind a different phosphorylated motif of known sequence, wherein the precise target sequences preferably bound by said antibodies has first been determined, thereby to detect at least one phosphoprotein comprising two or more of said precise target sequences; and    (c) utilizing a search program to search one or more protein database(s) for reported proteins having a sequence comprising the two or more precise target sequences detected in step (b) and having a mass and/or PI substantially matching that determined in step (a), thereby to positively identify at least one cellular phosphoprotein in said preparation as a reported protein of known sequence and mass and/or PI.    
     
     
         23 . A method for the positive identification of cellular phosphoproteins using motif-specific, context-independent antibodies coupled with database searching, said method comprising the steps of: 
 (a) fractionating a cellular preparation comprising a plurality of phosphopeptides by immunoaffinity isolation using at least one motif-specific, context-independent antibody that binds a phosphorylated motif of known sequence, wherein the mass of bound peptides is also determined; and    (b) utilizing a search program to search one or more protein database(s) for reported proteins having a sequence comprising said motif and having a mass substantially matching that determined in step (a), thereby to positively identify at least one cellular phosphoprotein in said preparation as a reported protein of known sequence and mass.